PROJECT SUMMARY In the last decade it has become clear that our understanding of hydrocephalus and the cerebrospinal fluid (CSF) circulation is incomplete. New information, such as 1) the description of CSF secretion and absorption within the brain, 2) the uncovering of oscillatory movements within the CSF, 3) the discovery of pathways that lead from CSF spaces into the brain parenchyma through paravascular channels, and 4) new pathways out via lymphatic drainage systems, has led to the understanding of a complex flow system which could ultimately better explain hydrocephalus and lead to more physiological treatments. While anatomic details guiding this flow of fluids and solutes are still unknown, their discovery would likely be a key to solving longstanding problems in understanding parenchymal changes in hydrocephalus and in achieving a treatment that allows a more physiological fluid regulation without over-and under-drainage. In this study we hypothesize a change in pattern and direction of net fluid flow between brain and CSF spaces that results in hydrocephalus and shunting failures. Anatomically this includes the reversal of fluid flow between the brain surface and ventricles. The use of our large-animal model of obstructive hydrocephalus, along with MRI tracking of Gadobutrol (604 Da) and fluorescent imaging of molecular tracers (Alexa Fluor 594 Hydrazide 758 Da, Dextran Lysine-fixable 3 kDa and 500 kDa) will allow the study of the activity of paravascular and interstitial flow in a controlled and clinically relevant manner, including changes with hydrocephalus and after variable CSF shunting. Canines will be distributed in three groups: normal control, obstructive hydrocephalus and shunted hydrocephalus. In Aim 1, we will assess the paravascular, interstitial and trans-ependymal flow in healthy canines, to understand the normal physiologic patterns of solute distribution around and inside the brain. In Aim 2, we will study the effects of obstructive hydrocephalus on these pathways of flow, investigating the signature that this pathology leaves on them, and their role in the onset of common symptoms and complications of hydrocephalus. In Aim 3, our focus will shift to CSF shunting, and the consequences that this has on the interstitial, paravascular and trans- ependymal systems. We hypothesize that interstitial flow reversals will be seen in hydrocephalus and shunting, and that parenchymal changes in hydrocephalus will be associated with changes in fluid and solute influx into brain from the ependyma and the paravascular pathways. This proposal, using multiple molecules and modalities, will be the first to demonstrate changes in solute movement in the canine brain in hydrocephalus and after treatment, opening a new avenue in understanding the physiopathology pathology of hydrocephalus in a clinically relevant canine model. Moreover, this approach can be extended to a variety of contexts and treatment issues, such as over- and undershunting, allowing a detailed histological evaluation and parallel clinical and experimental MR imaging. Finally, this platform can be a gold standard for developing noninvasive imaging for human investigation.